Panax notoginseng transcription factor gene PnHY5 and application thereof

Abstract

The invention discloses a panax notoginseng transcription factor gene PnHY5 and application thereof in increasing the total saponin content and monomer saponin content of panax notoginseng cells. The nucleotide sequence of the PnHY5 gene is as shown in SEQ ID NO: 1; according to the invention, functional genomics and metabolic engineering related technologies prove that the panax notoginseng PnHY5 transcription factor has a function of positively regulating and controlling the biosynthesis of notoginsenoside, and the panax notoginseng PnHY5 transcription factor gene is constructed on a plant expression vector and transferred into panax notoginseng cells for overexpression; the expression quantity of key enzyme genes in a synthetic route of the notoginsenoside is increased, and the biosynthesis of the notoginsenoside is promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of biosynthesis, and in particular relates to a notoginseng saponin biosynthesis-related transcription factor gene PnHY5 and a use thereof. Background Art

[0002] Panax notoginseng is a medicinal plant of the genus Panax in the Araliaceae family. Its roots and rhizomes are used as medicine and are a traditional precious Chinese medicinal material. Panax notoginseng has the dual effects of promoting blood circulation and stopping bleeding, and is an important component of the world-famous "Yunnan Baiyao". Notoginseng saponins are the main active ingredients of Panax notoginseng. More than 70 dammarane-type tetracyclic triterpenoid saponins have been isolated and identified from the root tuber, rhizome (cut end), stem, leaf and flower of Panax notoginseng, such as ginsenoside Rb1, Rg1, Rd, Re, etc. Most of these monomer saponins are dammarane 20(S)-protopanaxadiol type [20(S)-protopanaxatriol] and 20(S)-protopanaxatriol type [20(S)-protopanaxatriol], and no oleanolic acid-type saponins were found, which is significantly different from ginseng and American ginseng. Notoginseng saponins have the effects of dilating coronary arteries and peripheral blood vessels, increasing cerebral blood flow; they also have the effects of inhibiting platelet aggregation, reducing blood viscosity, and inhibiting thrombosis; at the same time, they have the effects of lowering blood lipids, resisting fatigue, resistant to hypoxia, and improving and enhancing macrophage function.

[0003] Panax notoginseng is a perennial medicinal plant with strict requirements on habitat, but the land area suitable for growing Panax notoginseng is limited; at the same time, Panax notoginseng suffers from serious diseases and insect pests during its growth, and the problems of secondary salinization and acidification of the soil are prominent, which leads to the need for "crop rotation" in Panax notoginseng planting. This long growth cycle and low land utilization rate have restricted the sustainable development of the Panax notoginseng industry. In recent years, the market for medicines with Panax notoginseng as a component has expanded rapidly, leading to an increase in demand for Panax notoginseng medicinal materials and a prominent contradiction between supply and demand. In view of the disadvantages of long artificial cultivation time and unclear chemical synthesis mechanism and route, the use of bioengineering technology and gene regulation methods to produce Panax notoginseng saponins has gradually become a research hotspot.

[0004] Light is the energy source for photosynthesis in plants and one of the most important environmental factors in the process of plant growth and development. In the process of plant growth and development, plants have evolved a set of complex light sensing pathways to adapt to changes in the light environment and respond to changes in the light environment. These light sensing pathways can respond to light intensity, light quality, light direction and day length. Among them, plants mainly rely on different photoreceptors to perceive light of different wavelengths and transmit light signals. The induction of light signals by photoreceptors can activate transcription factors of different families downstream of photoreceptors to transmit light signals. It has been reported that the transcription factor families that act downstream of photoreceptors include bZIP, bHLH, MYB, Zinc-finger, GATA and GT1. These transcription factors transmit light signals mainly by binding to light response elements on the promoters of downstream genes, such as G-box, GATA, MYB, etc., thereby initiating the transcription of downstream genes. HY5 belongs to the bZIP transcription factor family. The protein encoded by HY5 is a core light signal regulator in the light signal transduction pathway downstream of photoreceptors. It plays a very important role in the light signal transduction process and can directly regulate or interact with other transcription factors to initiate the expression of downstream genes. HY5 can directly bind to the binding sites of "ACGT-containing elements" on the promoter of genes, such as G-box (CACGTG), A-box (TACGTA), Z-box (ATACGGT) and C-box (GACGTC). G-box is widely present in the promoters of light-controlled genes and some key metabolic pathway enzyme genes. It has a highly conserved core motif CACGTG and is a universal regulatory element for plants to respond to external environmental stress. It is also the most clearly studied plant regulatory element in the field of plants. As a transcription factor, HY5 exerts transcriptional regulation by first binding to the cis-element containing "ACGT elements", and then controlling the expression of many target genes to respond to light signals, and ultimately regulating the physiological and biological processes of plants. HY5 can regulate many important links in the process of plant growth and development, such as cell elongation, cell proliferation, chloroplast development, pigment accumulation and nutrient assimilation. In recent years, studies have also reported the role of HY5 in other aspects, such as hormone signal transduction, secondary metabolite synthesis, plant defense and external environmental response. However, the role of HY5 transcription factor in metabolic regulation in Panax notoginseng is still unclear. Summary of the invention

[0005] The present invention provides a Panax notoginseng transcription factor gene PnHY5, which is cloned from Panax notoginseng. The present invention clarifies the purpose of the transcription factor, that is, it can increase the expression of key enzyme genes in the Panax notoginseng saponin synthesis metabolic pathway and increase the saponin content in Panax notoginseng cells.

[0006] The nucleotide sequence of the transcription factor gene PnHY5 cloned from Panax notoginseng cells of the present invention is shown in SEQ ID NO: 1. The full-length cDNA sequence of the gene is 495 bp and encodes a protein with an amino acid sequence shown in SEQ ID NO: 2.

[0007] The above-mentioned transcription factor genes can be used to positively regulate the biosynthesis of notoginseng saponins. The specific operation is as follows:

[0008] (1) Obtaining full-length cDNA of the gene: Using specific primers for amplifying PnHY5, total RNA was extracted from Panax notoginseng cells, and the coding region of PnHY5 was amplified by reverse transcription-polymerase chain reaction (RT-PCR). The amplified region was then ligated to the pMD18-T vector, and a clone containing the target gene was obtained by sequencing;

[0009] (2) Construction of plant expression vector and genetic transformation: The pMD18-T-PnHY5 plasmid was digested with restriction endonucleases XbaⅠ and PstⅠ, and the target gene fragment was obtained by gel recovery; the plant expression vector pCAMBIA2300S was digested with the same endonucleases, and the large vector fragment was obtained by gel recovery; the target gene fragment was connected with the pCAMBIA2300S vector fragment to construct the plant overexpression vector pCAMBIA2300S-PnHY5; the pCAMBIA2300S-PnHY5 plasmid was introduced into the Agrobacterium strain EHA105 by liquid nitrogen freeze-thaw method, and PnHY5 was introduced into Panax notoginseng cells by Agrobacterium-mediated genetic transformation to make it overexpressed, and positive transgenic cell lines were screened by antibiotic selection and fluorescence quantitative PCR;

[0010] (3) Detection of total saponin content in transgenic cell lines: Extract saponins from non-transgenic cells and transgenic cell lines of Panax notoginseng, analyze the difference in total saponin content between non-transgenic and transgenic cell lines, and screen out positive transgenic cell lines with increased total saponin content;

[0011] (4) Detection of the content of some important monomer saponins in transgenic cell lines: Saponin solutions of non-transgenic Panax notoginseng cells and transgenic cell lines with increased total saponin content were prepared, and the contents of some important monomer saponins in non-transgenic cell lines and transgenic cell lines were determined by HPLC. The differences in monomer saponin content between non-transgenic cells and transgenic cell lines were analyzed, and finally, positive transgenic cell lines with increased monomer saponin content were screened out.

[0012] The present invention uses in vitro cultured Panax notoginseng cells as the research object, clones the PnHY5 transcription factor gene, and performs functional identification on the transcription factor, thereby clarifying the position and role of the PnHY5 transcription factor in the biosynthesis process of notoginseng saponins, and providing a theoretical reference and basis for obtaining efficient and stable Panax notoginseng saponin synthesis regulation technology and the establishment of homologous or heterologous efficient expression systems.

[0013] The present invention provides a new method for increasing the content of saponins in Panax notoginseng. Panax notoginseng saponins can be synthesized more efficiently by utilizing bioengineering technology and gene regulation methods, thereby overcoming the shortcomings of long artificial cultivation period and unclear chemical synthesis mechanism and route. The transcription factor PnHY5 gene is introduced into Panax notoginseng cells for expression, thereby increasing the expression amount of key enzyme genes in the biosynthesis pathway of Panax notoginseng saponins and increasing the output of Panax notoginseng saponins, thereby providing a theoretical reference and scientific basis for large-scale industrial production of Panax notoginseng saponins. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is the amplification result of the full-length cDNA of PnHY5 in the present invention, wherein M is DL5000 DNA Marker, and 1 is the amplification product of the full-length cDNA of PnHY5;

[0015] Figure 2 The figure is a PCR test result of the bacterial solution of the plant expression vector pCAMBIA2300s-PnHY5 in the present invention transferred into Agrobacterium tumefaciens EHA105, in which the marker is DL5000 DNA Marker; 1-7 are bacterial solution samples; the positive control is the PCR product using the plasmid pMD18-T-PnHY5 as a template; the negative control is the PCR product using sterile water as a template;

[0016] Figure 3 This is the result of resistance gene detection in transgenic Panax notoginseng cells. The Maker in the figure is DL2000 DNA Maker; 1-3 are cDNA samples of transgenic Panax notoginseng cells; the positive control is the PCR product with plasmid pCAMBIA2300s-PnHY5 as template; the negative control is the cDNA of non-transgenic Panax notoginseng cells.

[0017] Figure 4 It is a graph of qRT-PCR result analysis in the present invention, showing the expression levels of FPS, HMGR and DS genes regulated by PnHY5 in the notoginseng saponin synthesis pathway in wild-type and transgenic cell lines, wherein WT is a wild-type cell line, and 1-3 are transgenic cell lines;

[0018] Figure 5 The total saponin content of Panax notoginseng cells in the present invention is determined, wherein WT is a wild-type cell line, and 1-3 is a transgenic cell line;

[0019] Figure 6 These are the results of determining the contents of some important monomer saponins in Panax notoginseng cells of the present invention, where WT is a wild-type cell line and 1-3 are transgenic cell lines. DETAILED DESCRIPTION

[0020] The present invention is further explained by examples below, but these examples do not limit the scope of protection of the present invention. The methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercial reagents or reagents prepared according to conventional methods unless otherwise specified;

[0021] Example 1: Cloning of PnHY5 gene fragment

[0022] Total RNA was extracted from Panax notoginseng cells (derived from Panax notoginseng callus), the Panax notoginseng cells were ground into powder with liquid nitrogen, and then transferred into a centrifuge tube, and the total RNA was extracted using the guanidine thiocyanate method; RNA with better quality was selected and the first chain of cDNA was synthesized using the GoScript reverse transcription system of Promega, Oligo(dT)15 1.0μL, Total RNA 5.0μg, and Nuclease-free Water were added to 10μL, mixed thoroughly and gently, and placed in a 70℃ water bath for 5min for pre-denaturation, and then immediately placed on ice for 5min, and then Nuclease-free Water 1.6μL, GoScript TM 5×Reaction Buffer 4.0μL, PCR Nucleotide Mix 1.0μL, MgCl2 (25mM) 2.0μL, Recombinant Ribonuclease Inhibitor 0.4μL, GoScript TMMix 1.0μL of Reverse Transcriptase, centrifuge briefly, anneal at 25℃ for 5min, extend in a 42℃ water bath for 90min, and finally, place in a 70℃ water bath for 15min to terminate the reverse transcriptase activity to obtain the first chain of Panax notoginseng cDNA. The synthesized first-strand cDNA was used as a template to amplify the target gene PnHY5. The upstream and downstream primers used were 5'-GGATCTTCCAGAGATTCTAGAATGCCAGACCCAGCAACAAG-3' and 5'-CTGCCGTTCGACGATCTGCAGTTATGTTTTGATAGGATGATTTGTGTAAA-3', respectively. The PCR reaction conditions were 94℃5min, 94℃30s, 60℃30s, 72℃30s (32 cycles), and 72℃7min. The reaction system (20μL) was 0.5μL cDNA, 2μL 10×Advantage2 PCR Buffer, 0.4μL 50×dNTP Mix (10mM each), 0.4μL forward primer (10μM), 0.4μL reverse primer (10μM), 0.4μL Advantage 2PCR Polymerase Mix, and 15.9μL PCR-Grade After PCR, take 5 μL for agarose gel electrophoresis to detect the specificity and size of the amplified product and amplify the target gene such as Figure 1 shown.

[0023] The kit pMD18-T Vector System I (Promega, USA) was used for TA cloning. The reaction system and operation process were as follows: 8 μL of PCR product was added in sequence with 1 μL of pMD18-T Vector (50 ng / μL) and 1 μL of 2×Ligationsolution I, mixed well and placed at 16°C overnight for reaction, and the ligation product was transformed into Escherichia coli DH5α by heat shock transformation. Positive clones were screened using LB solid medium containing ampicillin (Amp), and several single colonies were selected. After shaking the bacteria, they were identified using specific primers for amplifying PnHY5, and the successfully constructed plasmid pMD18-T-PnHY5 was obtained.

[0024] Example 2: Plant overexpression vector construction

[0025] The Escherichia coli plasmid pMD18-T-PnHY5 with the PnHY5 gene inserted and the plant expression vector pCAMBIA2300s plasmid were extracted using the SanPrep column plasmid DNA mini-extraction kit (Shanghai Sangon Biotechnology Co., Ltd., China). 1 μL was used for agarose gel electrophoresis to detect the integrity and concentration of the extracted plasmids. Plasmids pMD18-T-PnHY5 and pCAMBIA2300s were double-digested with restriction endonucleases XbaⅠ (TaKaRa, Japan) and PstⅠ (TaKaRa, Japan) (50 μL system), respectively. The reaction system and operation process were as follows: 20 μL of pMD18-T-PnHY5 and pCAMBIA2300s plasmids were added with 7.5 μL 10×K buffer, 2.5 μL XbaⅠ, 2.5 μL PstⅠ, and 17.5 μL ddH2O in sequence, mixed and centrifuged for a short time, and placed at 37℃ for enzyme digestion for 3 h. All enzyme digestion products were spotted on agarose gel for electrophoresis, and then the PnHY5 fragment and the large fragment of the pCAMBIA2300s vector were gel-recovered, respectively. The entire process used the SanPrep column DNA gel recovery kit (Sangon Biotech (Shanghai) Co., Ltd., China). Take 1.0 μL of the recovered product and detect the size and concentration of the recovered fragments by agarose gel electrophoresis, and store it at -20°C for future use.

[0026] The recovered PnHY5 DNA fragment and pCAMBIA2300s vector fragment were connected by T4 DNA Ligase (TaKaRa, Japan). The reaction system (10 μL) and operation process were as follows: 6 μL PnHY5 DNA fragment was added to 2 μL pCAMBIA2300s vector DNA, 1 μL 10×T4 DNA Ligase Buffer, and 1 μL T4 DNA Ligase in turn, mixed and centrifuged for a short time, and then reacted in a water bath at 16°C overnight. Then, the connection product was transformed into Escherichia coli DH5α by heat shock transformation, and positive clones were screened with solid culture medium containing 50 mg / L kanamycin (kanamycin, Kana). Single colonies were selected and shaken, and PCR was performed with specific primers for amplifying PnHY5 using the bacterial liquid as a template to select clones that successfully connected PnHY5 and pCAMBIA2300s. If the detected strain was positive, glycerol was added and stored at -80°C for later use.

[0027] Extract and purify the pCAMBIA2300s-PnHY5 plasmid in the above Escherichia coli. Then, the plant expression vector pCAMBIA2300s-PnHY5 constructed above was transferred into Agrobacterium tumefaciens EHA105 competent cells by liquid nitrogen freeze-thaw method. The operation steps are as follows: take 2μg pCAMBIA2300s-PnHY5 plasmid and add it to a centrifuge tube containing 100μL competent cells, mix gently and place on ice for 5min, then transfer to liquid nitrogen and freeze for 5min, then quickly place in a 37℃ water bath for 5min, then immediately place on ice for 2min, add 800μL LB liquid culture medium and shake culture at 28℃ for 4h. Apply the activated Agrobacterium to LB solid culture medium containing 50mg / L Kana and 20mg / L Rif, and culture statically at 28℃. Single colonies were selected and shaken, and PCR was performed using specific primers for amplifying PnHY5 to detect whether pCAMBIA2300s-PnHY5 was transferred into Agrobacterium. For positive clones, glycerol was added and stored at -80°C for later use. The PCR analysis results for detecting the transfer of pCAMBIA2300s-PnHY5 into Agrobacterium EHA105 are shown in Figure 2 , and the plant overexpression vector pCAMBIA2300s-PnHY5 was successfully constructed.

[0028] Example 3: Agrobacterium-mediated plant genetic transformation and transgenic plant screening

[0029] Take out the Agrobacterium EHA105 strain containing the pCAMBIA2300s-PnHY5 plasmid from the -80℃ freezer, inoculate it into 5mL LB liquid medium containing 50mg / L Kana and 25mg / L rifampicin, and culture it at 28℃ until it becomes turbid. Pipette 1mL of the turbid bacterial liquid onto the LB solid medium containing 50mg / L Kana, and culture it at 28℃ for 48h. Scrape off the Agrobacterium on the LB solid medium and inoculate an appropriate amount into the MGL liquid medium, add 50mg / L acetosyringone, and shake culture at 28℃ until the OD 600 When the pH value reached 0.6, shaking was stopped and the obtained bacterial solution was used to infect Panax notoginseng cells.

[0030] The Panax notoginseng cells with good growth status were inoculated into the MS pre-culture medium (50mg / L acetosyringone) for pre-culture for 3 days. After the pre-culture was completed, the cells were completely immersed in the above-mentioned Agrobacterium bacterial solution for shaking culture. After the infection was completed, the bacterial solution was removed, the bacterial solution on the cell surface was absorbed with sterile filter paper, and then the cells were inoculated into the MS co-culture medium (containing 50mg / L acetosyringone) for co-culture for 3 days. After co-culture, the Panax notoginseng cells were carefully transferred to the sterilization medium (cephalosporin was added to the Panax notoginseng cell culture medium, and the Panax notoginseng culture medium was MS culture medium + 2,4-D 4mg / L + KT 1mg / L + 6-BA 0.5mg / L, pH5.6). The cephalosporin content in the sterilization medium was 200mg / L, 100mg / L, and 50mg / L, respectively. In order from high to low concentration, they were placed on the sterilization medium for 3 days until there was no Agrobacterium growth on the surface of the culture medium. After 9 days of sterilization and culture, the Panax notoginseng cells are transferred to a screening medium (neomycin is added to the Panax notoginseng cell culture medium to a final concentration of 50 mg / L), and subcultured approximately every 35 days. After 4-5 subcultures and screening, a transgenic Panax notoginseng cell line can be obtained.

[0031] The genomic DNA of transgenic Panax notoginseng cell lines was extracted by the modified CTAB method, and the upstream and downstream primers npt II were designed according to the kanamycin npt II resistance gene sequence on the T-DNA in the pCAMBIA2300s vector. F (5'-CTCTGATGCCGCCGTGTT-3') and npt R (5'-CCCTGATGCTCTTCGTCCA-3'), PCR detection was performed using the genomic DNA of the Panax notoginseng cells as a template to screen for positive transgenic Panax notoginseng cells.

[0032] Results Figure 3 In the figure, a specific band of about 0.4kb was amplified, which was consistent with the expected size, indicating that exogenous DNA had been introduced into the three Panax notoginseng transgenic cell lines and integrated into the genomic DNA for stable inheritance. It was preliminarily determined that the PnHY5 gene transgenic Panax notoginseng cell line was obtained.

[0033] Example 4: Effect of PnHY5 gene overexpression on the expression of key enzyme genes FPS, HMGR and DS in the notoginseng saponin synthesis pathway

[0034] Panax notoginseng transgenic cell lines and Panax notoginseng wild-type cell lines of about 25 days old and in good growth condition were selected, RNA was extracted respectively, and the first-chain cDNA was synthesized using Promega's GoScript reverse transcription system. The reaction system and operation process were as follows: RNA with good quality was selected and the first-chain cDNA was synthesized using Promega's GoScript reverse transcription system, Oligo(dT)151.0μL, Total RNA 5.0μg, and Nuclease-free Water were added to 10μL and mixed thoroughly and gently, and placed in a 70℃ water bath for 5min for pre-denaturation, and then immediately placed on ice for 5min, and then the following reagents were added: Nuclease-free Water 1.6μL, GoScriptTM5×Reaction Buffer 4.0μL, PCR NucleotideMix 1.0μL, MgCl2(25mM)2.0μL, Recombinant Ribonuclease Inhibitor 0.4μL, GoScript TM Mix 1.0 μL of Reverse Transcriptase, centrifuge briefly, anneal at 25°C for 5 min, extend in a 42°C water bath for 90 min, and finally, place in a 70°C water bath for 15 min to terminate the reverse transcriptase activity to obtain the first chain of Panax notoginseng cDNA.

[0035] Using the cDNA as a template, primers were designed based on the Panax notoginseng GAPDH gene (accession number: KF815711.1), farnesyl pyrophosphate synthase (FPS) gene (accession number: DQ059550.1), 3-hydroxy-3-methylglutaryl coenzyme A reductase (3-hydroxy-3-methylglutaryl coenzyme A reductase, HMGR) gene (accession number: KJ578757.1) and dammarenediol synthase (Dammarenediol-II synthase, DS) gene (accession number: KC953035.1). The operation was performed according to the qPCR Master Mix instruction manual. The primer sequence used was GAPDH F :5'-CTACCAACTGTCTTGCTCCCCT-3';GAPDH R :5'-TGATGCAGTCTTCCACCTCTC-3';FPS F :5'-CGGATGCTGGACTATAATGTG-3';FPS R:5'-ATTTACGGCAATCATACCAACC-3'; HM GR F :5'-GGCAGGACCCAGCACAAAATA-3';HMGR R :5'-ACACCCAGAAGGTTCAAGCAA-3'DS F :5'-TATGAGTGGGAAGGGTGC-3'; DS R :5'-TGGCGATAATTGCTTGAGTA-3'. The specific reaction system and operation process are as follows: add 20ng cDNA, 25μL qPCR Master Mix (2×) and 0.2μL qPCRPrimers (GAPDH F / GAPDH R ,FPS F / FPS R , HMGR F / HMGR R , DS F / DS R , 10mM), and filled up to 50μL with Nuclease-Free Water; after vortexing the reaction system, centrifuge it to collect it at the bottom of the tube, and then place it in a fluorescent quantitative PCR instrument for reaction. The two-step method was used for fluorescent quantitative PCR. The reaction parameters were as follows: hot start at 95℃ for 2min; denaturation at 95℃ for 15s, annealing / extension at 60℃ for 1min, for a total of 45 cycles, and each gene corresponding to each sample was detected twice.

[0036] qRT-PCR results showed that in PnHY5 transgenic Panax notoginseng cells, the expression levels of FPS, HMGR and DS genes were significantly higher than those of wild type ( Figure 4 ), indicating that PnHY5, as a transcription factor, can promote the expression of key enzyme genes FPS, HMGR and DS in the notoginseng saponin biosynthesis pathway.

[0037] Example 5: Effect of PnHY5 gene overexpression on the synthesis of total saponins of Panax notoginseng

[0038] Select transgenic cell lines and wild-type cell lines that have grown for about 35 days, place them in a clean 100mL conical flask, add 20mL of methanol solution and soak overnight, then treat with ultrasound at room temperature for 1h, filter, collect the filtrate, concentrate the filtrate and dissolve it with methanol, fix the volume to 25mL, and obtain a crude extract; dry the residue at 50℃ to constant weight and weigh it. Accurately draw 5mL of crude extract, place it in a 50mL beaker and evaporate it in a water bath; after evaporation, dissolve it in 4 times the volume of distilled water, filter it after sufficient dissolution, and transfer all the filtrate to the treated Hsp100 macroporous resin column, first use 2 column volumes of distilled water to slowly wash away impurities such as sugars. Use the Molish reaction to detect whether the sugar impurities are completely removed. If the result is positive, continue to wash with distilled water until negative, then elute with 75% ethanol solution for 2 column volumes, collect the alcohol solution, evaporate it in a water bath, dissolve the residue with methanol solution, and fix the volume to 25mL.

[0039] Accurately pipette 150 μL of the above sample into a 10 mL test tube (set 3 replicates), evaporate the solvent, add 0.2 mL of freshly prepared 5% vanillin-glacial acetic acid solution and 0.8 mL of perchloric acid, mix well and heat in a 60°C water bath for 15 min, immediately cool with ice water, add 5 mL of glacial acetic acid, mix well and let stand for 10 min, measure the absorbance at 554 nm, and calculate the PNS content with reference to the standard curve. The results showed that the total saponin content in the PnHY5 gene-transfected Panax notoginseng cells was higher than that in the wild-type cells ( Figure 5 ), combined with the qRT-PCR results, indicated that the PnHY5 transcription factor was involved in the regulation of the synthesis and metabolism of notoginseng saponins and contributed to the increase of saponin production.

[0040] Example 6: Effect of PnHY5 gene overexpression on the synthesis of Panax notoginseng monomer saponins

[0041] The content of some important monomer saponins (Rb1, Rg1, Rd, Re) in Panax notoginseng cell lines was determined by HPLC. The HPLC conditions were as follows: HPLC, Waters symmertry C18 column (4.6×250mm, 5μm), acetonitrile (A): water (B) as mobile phase for linear gradient elution (v / v), detection column temperature of 30°C, detection wavelength of 203nm, and flow rate set to 1.0mL / min.

[0042] Accurately weigh appropriate amounts of monomer saponin Rb1, Rg1, Rd, and Re standards, add 1 mL of methanol solution, and prepare standard mixed solutions with concentrations of 340, 300, 260, 320, 280, and 300 μg / mL, respectively. Pipette 4, 6, 8, 10, 15, 20, 25, and 30 μL of the mixed standard solution and inject it into the high performance liquid chromatograph. With the injection volume (μg) as the abscissa (x) and the area of ​​the chromatographic peak as the ordinate (y), draw the linear regression equation of each monomer saponin.

[0043] Weigh 0.1g of non-transgenic and transgenic Panax notoginseng cell powder with improved total saponin content respectively into a clean 50mL conical flask, add 10mL of 70% methanol solution to each, soak overnight and treat with an ultrasonic crusher for 90min (60w, 4s / 5s). After the end of the ultrasound, remove the filter residue and keep the filtrate, and place the filtrate in a 50℃ oven overnight for drying. Add 10mL of distilled water to dissolve the dried residue, then extract 2-3 times with the same volume of water-saturated n-butanol (do not use n-butanol that has not been saturated with water), and place the final collected extract in a 50℃ oven overnight for drying. Use an appropriate amount of 100% methanol to dissolve the dried residue, then make the solution dilute to 5mL, mix well and filter through a 0.45μm filter membrane, and use high performance liquid chromatography to determine the content of some important monomer saponins in the saponin solution. The results showed that the four monomer saponins Rb1, Rg1, Rd and Re were detected in both non-transgenic Panax notoginseng cells and transgenic Panax notoginseng cell lines. Compared with non-transgenic cell lines, the four monomer saponins increased in transgenic cell lines to varying degrees ( Figure 6 ).

Claims

1. A Panax notoginseng transcription factor gene PnHY5, whose nucleotide sequence is shown in SEQ ID NO:

1.

2. Use of the Panax notoginseng transcription factor gene PnHY5 described in claim 1 in increasing the total saponin content and monomer saponin content of Panax notoginseng cells.

Images